Antimalarial efficacy and toxicological assessment of medicinal plant ingredients of Prabchompoothaweep remedy as a candidate for antimalarial drug development

Background Drug resistance exists in almost all antimalarial drugs currently in use, leading to an urgent need to identify new antimalarial drugs. Medicinal plant use is an alternative approach to antimalarial chemotherapy. This study aimed to explore potent medicinal plants from Prabchompoothaweep remedy for antimalarial drug development. Methods Forty-eight crude extracts from Prabchompoothaweep remedy and its 23 plants ingredients were investigated in vitro for antimalarial properties using Plasmodium lactate dehydrogenase (pLDH) enzyme against Plasmodium falciparum K1 strain and toxicity effects were evaluated in Vero cells. The plant with promising antimalarial activity was further investigated using gas chromatography-mass spectrometry (GC-MS) to identify phytochemicals. Antimalarial activity in mice was evaluated using a four-day suppressive test against Plasmodium berghei ANKA at dose of 200, 400, and 600 mg/kg body weight, and acute toxicity was analyzed. Results Of the 48 crude extracts, 13 (27.08%) showed high antimalarial activity against the K1 strain of P. falciparum (IC50 <  10 μg/ml) and 9 extracts (18.75%) were moderately active (IC50 = 11–50 μg/ml). Additionally, the ethanolic extract of Prabchompoothaweep remedy showed moderate antimalarial activity against the K1 strain of P. falciparum (IC50 = 14.13 μg/ml). Based on in vitro antimalarial and toxicity results, antimalarial activity of the aqueous fruit extract of Terminalia arjuna (IC50 = 4.05 μg/ml and CC50 = 219.6 μg/ml) was further studied in mice. GC-MS analysis of T. arjuna extract identified 22 compounds. The most abundant compounds were pyrogallol, gallic acid, shikimic acid, oleamide, 5-hydroxymethylfurfural, 1,1-diethoxy-ethane, quinic acid, and furfural. Analysis of the four-day suppressive test indicated that T. arjuna extract at dose of 200, 400, and 600 mg/kg body weight significantly suppressed the Plasmodium parasites by 28.33, 45.77, and 67.95%, respectively. In the acute toxicity study, T. arjuna extract was non-toxic at 2000 mg/kg body weight. Conclusions The aqueous fruit extract of T. arjuna exerts antimalarial activity against Plasmodium parasites found in humans (P. falciparum K1) and mice (P. berghei ANKA). Acute toxicity studies showed that T. arjuna extract did not show any lethality or adverse effects up to a dose of 2000 mg/kg.


Plant materials
Twenty-three plant ingredients from the Prabchompoothaweep remedy were purchased from a traditional Thai drug store in the Nakhon Si Thammarat Province, Thailand ( Table 1). The use of plant materials complied with the relevant guidelines and regulations of the Plant Varieties Protection, Department of Agriculture, Ministry of Agriculture and Cooperatives, Thailand. The plant species were identified by Assoc. Prof. Tanomjit Supavita, School of Pharmacy at Walailak University. Voucher herbarium specimens were deposited in the School of Medicine, Walailak University (Table 1).

Plant extraction
All plant samples were cleaned with distilled water to remove dirt and dried at 60 °C in a hot air oven for 72 h. The plant samples were then cut into small pieces and weighed into portions of 60 g. Each plant was extracted using ethanol and distilled water. Ethanol was selected as a solvent due to it can dissolve most slightly non-polar and slightly polar molecules. Distilled water was used as the solvent to be related to the usage almost plants as Thai folk medicines. For ethanolic extraction, the plant samples (60 g) were macerated in 600 ml of 80% ethanol at 25-30 °C for 72 h and this procedure was repeated three times. The aqueous extract was obtained using the decoction method and 60 g of each plant was extracted three times by mixing with 600 ml of distilled water and boiled at 90-100 °C for 30 min. The resulting extract in each method was filtered through Whatman No. 1 filter paper, evaporated in a rotary evaporator (N-1200B, EYELA Co., Ltd., Shanghai, China) at 60 °C, and lyophilized to dryness using a freeze-dryer (Gamma 2-16 LSCplus, Martin Christ, Osterode am Harz, Germany). The crude extracts were collected and stored at 4 °C until use.

Phytochemical analysis
All the extracts were subjected to standard phytochemical analyses to determine the presence of flavonoids, terpenoids, alkaloids, tannins, anthraquinone, cardiac glycosides, saponins, and coumarins, as previously described with some modifications [13,14].

In vitro cultivation of Plasmodium falciparum
To investigate in vitro antimalarial activity, P. falciparum K1 strain was cultured as previously described with minor modifications [15]. The Plasmodium parasite was cultured in uninfected O + red blood cells (RBCs) as host cells and maintained in complete medium (RPMI-1640) containing 2 mg/ml sodium bicarbonate, 10 μg/ml hypoxanthine (Sigma-Aldrich, New Delhi, India), 4.8 mg/ ml HEPES (HiMedia, Mumbai, India), 0.5% Albumax II (Gibco, Waltham, MA, USA), and 2.5 μg/ml gentamicin (Sigma-Aldrich). The culture flasks were incubated at 37 °C and 5% CO 2 . The percentage of parasitemia was monitored daily using a light microscope.

In vitro antimalarial activity assay
Prabchompoothaweep remedy and its plant ingredient extracts were tested for their antimalarial activity using an in vitro Plasmodium lactate dehydrogenase (pLDH) assay [16]. In this assay plates containing 2% parasite cultures were incubated with crude extract at final concentrations between 0.3-2500 mg/ml for 72 h at 37 °C in a CO 2 incubator. Artesunate (0.3-2500 mg/ml) (Sigma-Aldrich) and dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) were added to each well as positive and negative controls, respectively. Non-infected RBCs were used as blank controls. After 72 h of incubation, the plates were frozen at − 20 °C and thawed at 37 °C three times. The supernatant from each well was transferred to a new microplate containing the Malstat reagent (Sigma-Aldrich). Nitroblue tetrazolium/phenazine ethosulfate solution (Sigma-Aldrich) was added to the plate and cultured in the dark for 60 min. Next, 5% acetic acid (Merck) was added to each well to stop the reaction. The absorbance at 650 nm was measured using a microplate reader. Each sample was tested in triplicates. Finally, a log doseresponse curve was generated and used to determine the percent inhibition and half-maximal inhibitory concentration (IC 50 ).

In vitro cytotoxicity assay
Vero cells (1 × 10 5 /well) were plated in 200 μl of complete medium per well in 96-well plates. After cell attachment, the plant extracts were added and incubated at 37 °C for 24 h. Concentrations of plant extract varied from 0 to 80 μg/ml. The culture medium was then replaced with 100 μl of fresh medium/well containing 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) per well and incubated at 37 °C for 3 h. DMSO was then added to each well and incubated for another 20 min at room temperature in the dark. Lastly, the absorbance at 560 and 670 nm was measured using a microplate reader. All experiments were repeated thrice. The 50% cytotoxic concentration (CC 50 ) of the extracts was determined by dose-response curve analysis [17].

Selectivity index
A selectivity index (SI), which is the ratio between cytotoxic and antimalarial activities [18], was calculated for each extract according to the following formula:

GC-MS analysis
The relative quantities of the phytochemicals present in the extracts were determined using gas chromatography with a 7000C Triple Quadrupole GC/MS (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5MS column (30 m × 0.25 mm; 0.25 μm). Spectroscopic detection by GC-MS involves an electron ionization system that utilizes high energy electrons of 70 eV, ion source temperature of 250 °C, and mass scanning range of 33-600 amu in full scan. Pure helium gas (99.99%) was used as the carrier gas at a constant flow rate of 1 ml/min. The injector temperature was maintained at a constant of 250 °C, and the oven temperature was programmed as follows: 60 °C for 2 min, 150 °C at an increasing rate of 10 °C/min, and finally, 300 °C at an increasing rate of 5 °C/ min. The sample (1 μl) in ethanol was injected in the split mode at a split ratio of 20:1, respectively. The compounds in the test samples were identified by comparing their retention times and mass spectra with those in the spectral database of the National Institute of Standards and Technology (NIST2011) structural library. Only peaks with 80% similarity and above with the NIST libraries were selected and identified.

Four-day suppressive test (Peter's test)
The four-day suppressive test was used to measure the schizonticidal activity of the aqueous extract of T. arjuna against P. berghei ANKA-infected ICR mice. The method was performed as previously described with minor modifications [19,20]. Briefly, male ICR mice were randomly divided into five groups of five animals. Twenty-five mice were injected with 1 × 10 7 RBCs infected with P. berghei ANKA via intraperitoneal injection [20]. The treatment started 4 h following inoculation. In the extract treatment groups, the animals received daily oral doses of 200, 400, or 600 mg/kg body weight aqueous extract of T. arjuna in 200 μl of 7% Tween 80 solution. The dosage was selected with increasing as low, moderate and high doses of crude extract with 200, 400, and 600 mg/kg body weight according to previous studies [20][21][22]. The negative control group received 200 μl of 7% Tween 80 solution, while the positive control group was administered 6 mg artesunate/kg body weight orally per day. The mice were administered each substance daily for 4 days (at 4, 24, 48, and 72 h after inoculation). On the fifth day, the percentage of parasitemia was determined using Giemsa staining. Percent inhibition was calculated using the following formula [23]:

Determination of mean survival time (MST)
MST was determined as described by Chaniad et al. [20]. Twenty-five mice were used in the four-day suppressive test and fed ad libitum. Mouse mortality was monitored daily until day 30 after parasite inoculation. Any deaths in the treatment and control groups that occurred during the follow-up period were recorded. The MST for each group was calculated using the following formula [23]:

Acute toxicity test
The crude aqueous extract of T. arjuna was assessed for toxicity in non-infected ICR mice aged 6-8 weeks old and weighing 25-30 g according to the standard guidelines of the Organization for Economic Cooperation and Development [24]. Fifteen mice were randomly divided into three groups of five mice each: mice treated with and creatinine [Cr]) using an AU480 chemistry analyzer (Beckman Coulter, Brea, CA, USA). Furthermore, liver and kidney tissues were removed and fixed in formalin for histopathological examination.

Histopathology
Histopathological examination of the liver and kidney tissues was performed according to previously described histological procedures [25,26]. All tissue were fixed in 10% buffered formalin, then dehydrated using a gradient series of ethanol solutions, rinsed three times with xylene, and placed in a mold containing paraffin. The paraffin blocks were then serially sectioned at 5 μm thickness, transferred to glass slides, and stained with hematoxylin and eosin solution. To evaluate histopathological changes, the stained slides were observed using a light microscope by two independent researchers blinded to the experimental groups.

Statistical analysis
The results are presented as mean ± standard error of the mean (SEM). IBM SPSS Statistics version 23.0 software was used for the statistical analysis. The Kolmogorov-Smirnov goodness-of-fit test was used to test the normal distribution. The statistical significance of parasitemia inhibition was analyzed using one-way analysis of variance, followed by Tukey's multiple comparison test. Statistical significance was set at p-value less than 0.05 (p ≤ 0.05).

Extraction of plant materials
The percentage of crude extract yield (%yield) as shown in Table 2

Phytochemical screening
Phytochemical analysis of each plant component in Prabchompoothaweep remedy revealed the presence of flavonoids, terpenoids, alkaloids, tannins, saponins, and coumarins, whereas anthraquinones and cardiac glycosides were not detected in any of the extracts (Table 3). Moreover, the ethanolic extract of the remedy contained terpenoids, alkaloids, tannins, and coumarins, whereas the aqueous extract of the remedy contained flavonoids, terpenoids, alkaloids, tannins, saponins, and coumarins (Table 3).

In vitro antimalarial activity
The in vitro antimalarial activity of Prabchompoothaweep remedy and its ingredients is shown in

In vitro cytotoxicity
The evaluation of in vitro toxicity in Vero cells is shown in Table 4. A non-toxic effect is defined as a CC 50 value greater than 50 μg/ml [28]. Therefore, all extracts were non-toxic to Vero cells with CC 50  Of the total 48 extracts, the aqueous extract of T. arjuna was found in the top five extracts with an antimalarial effect and was non-toxic to Vero cells. This extract showed promising antimalarial activity (IC 50 = 4.05 μg/ml) against the K1 strain of P. falciparum and no cytotoxic effect against Vero cells (CC 50 > 200 μg/ml). Based on the high antimalarial activity and SI values obtained for the aqueous fruit extract of T. arjuna and no previous report of its antimalarial activity, the in vivo antimalarial activity and acute toxicity of this extract was further evaluated in mice.

GC-MS analysis of ethanolic aqueous fruit extract of T. arjuna
The GC-MS chromatograms of the fruit extract of T. arjuna are shown in Fig. 1. The mass spectra of the phytochemical compounds were compared with those in the spectral database of known compounds in the NIST library. Twenty-two compounds were identified and characterized ( Table 5). The most abundant compound was pyrogallol with a retention time of 11.690 min (40.69%), followed by gallic acid (9.87%), shikimic acid (7.19%), oleamide (6.11%), and 5-hydroxymethylfurfural (5.72%), 1,1-diethoxy-ethane (3.11%), quinic acid (2.44%) and furfural (1.08%). Other compounds were present at concentrations below 1%. Chemical structure of eight compounds with the peak area greater than 1% was illustrated in Fig.2. Particularly, 5-hydroxymethylfurfural, compounds 8 with a retention time of 9.631 and maltol, compound 9 with a retention time of 9.944 have the same formula as C 6 H 6 O 3 but the spectrum patterns are different (Fig. 3). Interestingly, the identified compounds, i.e., benzenetriol (pyrogallol), trihydroxybenzoic acid (gallic acid), shikimic acid, and cinnamic acid, are interrelated via the biosynthesis pathway (Fig.4).

Four-day suppressive test
The four-day suppressive test showed that mice treated with aqueous fruit extract of T. arjuna at concentrations of 200, 400, and 600 mg/kg presented significantly (p < 0.001) lower percentages of parasitemia (24.58, 18.60, and 10.99%, respectively) compared with that in the negative control group (34.30%). Mice treated with aqueous fruit extracts of T. arjuna exhibited parasite suppression rates in a dose-dependent manner, with a maximum activity of 67.95%, followed by 45.77 and 28.33% at doses of 600, 400   and 200 mg/ml, respectively ( Table 6). The survival time of all mice was also assessed over a 30-d period, as shown in Table 6. The MST of the extract-treated groups was dosedependent. Extract doses of 200, 400, and 600 mg/kg body weight significantly (p < 0.05) prolonged the survival time by 13.00, 16.00, and 17.40 d, respectively, compared with that in the negative control mice (8.60 d). Additionally, compared with the 200 mg/kg extract-treated group, the mean survival durations of the 400 and 600 mg/kg extracttreated groups were significantly extended (Table 6).

In vivo acute toxicity biochemical tests
All mice treated with 2000 mg/kg aqueous fruit extract of T. arjuna revealed no gross physical or behavioral    changes, including lacrimation, altered feeding activities, vomiting, diarrhea, abnormal secretion, abnormal sleep, excitement, and hair erection for 24 h, and no mortality occurred during the 14-d follow-up period. Therefore, the lethal dose of the extract was greater than 2000 mg/kg body weight. To determine the effects of the aqueous fruit extract of T. arjuna on the liver and kidney, plasma biomarkers of liver and kidney functions were examined. The findings demonstrated that the mean levels of ALT, ALP, BUN, and Cr in the mice treated with 2000 mg/kg T. arjuna extract did not significantly differ from those in the 7% Tween 80 and untreated control groups (Table 7). However, the mean levels of AST in mice treated with 2000 mg/kg T. arjuna extract were significantly higher than those in the 7% Tween 80 group (p < 0.05).

Histopathological changes
Histopathological examination revealed that the mice treated with 2000 mg/kg T. arjuna extract exhibited normal histopathological features in both liver and kidney tissues compared with those in the negative control group (Fig. 5). Therefore, the aqueous fruit extract of T. arjuna at a dose of 2000 mg/kg body weight did not have acute hepatotoxic or nephrotoxic effects.

Discussion
Prabchompoothaweep remedy has long been used in Thai traditional medicine to relieve the common cold, hay fever, allergic rhinitis, and upper respiratory tract disease [5,6]. The aqueous and ethanolic extracts of all plant ingredients from the Prabchompoothaweep remedy were investigated for the presence of phytochemical constituents and then for antimalarial properties against P. falciparum K1 strain and cytotoxicity in Vero cells. In our in vitro study, the extracts of Prabchompoothaweep remedy and its plant ingredients were tested using enzymatic detection of the pLDH enzyme. The toxicity of the extract was next examined in Vero cells. According to the cell cytotoxicity classification, the CC 50 value was used to define the potency of cytotoxicity. A non-toxic effect is classified as a CC 50 value greater than 50 μg/ml [28]. The extracts exhibited varying degrees of antimalarial activity. Among the 48 crude extracts tested in the present study, the aqueous flower extract of S. aromaticum showed the highest antimalarial activity with the lowest IC 50  aromaticum, it also known as clove. It has been reported that methanolic extract of this plant possesses slightly antimalarial effect in mice infected with P. berghei [32]. For P. chaba, piperine which is the major isolated constituent of this plant has been reported to exhibit the antimalarial effect against both chloroquine-sensitive and   previous reports of the methanolic extract of T. arjuna bark exhibiting a non-cytotoxic effect on human peripheral blood mononuclear cells [34]. Therefore, the aqueous extract of T. arjuna was selected for further in vivo experiments.
The SI value is a crucial parameter for determining whether further work on an extract is warranted [35]. When the SI results are greater than 10, the extract is considered potentially safe in terms of cytotoxicity parameters [36]. Therefore, the aqueous T. arjuna extract with an SI value greater than 54.22 suppressed P. falciparum infection without acute toxic effects in mammalian cells. To confirm the in vitro antiplasmodial results, the antimalarial properties and toxic effects of this plant extract were further tested in an animal model.
Malaria-infected mice treated with the aqueous T. arjuna extract showed a significant dose-dependent decrease in the number of Plasmodium parasites. Furthermore, MST is an important parameter for evaluating the antimalarial activity of plant extracts. T. arjuna extract prolonged the survival of P. berghei-infected mice in a dose-dependent manner. This may be because secondary metabolites that exhibit anti-inflammatory and antioxidant functions were present and prevented the overall pathologic effect of the parasite in the infected mice [37,38]. Since, malaria is a highly inflammatory and oxidative disease. During the blood stage of malaria infection, in response to the presence of the parasite, the host's immune system produces proinflammatory cytokines, including IL-6, IL-8, IFN-γ, and TNF which play a pivotal role in controlling the growth of the parasite and its elimination [39]. In addition, during the blood stage of infection, the level of oxidative stress in plasma is increased, since it contributes to the elimination of invading pathogens, but also causes molecular damage in the host [40]. The potential of T. arjuna extract that exerts anti-inflammatory and antioxidant effects was supported by a previous report [41]. It inhibited the lipid peroxidation, maintained endogenous antioxidant enzyme activities and decreasing cytokine levels leading to decelerate the disease progression. Therefore, the antimalarial effect of the T. arjuna extract may be possessed by anti-inflammatory and antioxidant properties.
To confirm the safety of the extract, mice received a single dose of 2000 mg/kg aqueous T. arjuna extract. There were no visible signs or symptoms of toxicity or mortality in the mice. This indicated that the lethal dose of 50% was greater than 2000 mg/kg. Our study is in accordance with previous studies in which oral administration of methanolic extract of T. arjuna bark at various concentrations of 250-2000 mg/kg body weight did not show any adverse signs of toxicity or mortality in acute toxicity study in mice [34].
Biochemical analysis of liver and kidney functions plays an important role in evaluating the toxicological effects of xenobiotics [42,43]. The plasma levels of ALT and ALP in mice treated with aqueous T. arjuna extracts were not significantly different compared with the untreated control and 7% Tween 80 groups. Regarding the kidney function test, BUN and Cr levels were not significantly different between the groups. Histopathological analysis of the liver and kidneys revealed normal features compared with those in healthy mice.
Phytochemical analysis of Prabchompoothaweep remedy showed a diversity of phytochemical constituents, including flavonoids, terpenoids, alkaloids, tannins, saponins, and coumarins. These secondary metabolites prevent the generation of free radicals and block protein synthesis in the Plasmodium parasite [5,[44][45][46][47]. Saponins may also modulate the immune system of infected mice [22]. Moreover, saponins are amphiphilic nature and can complex with cholesterol in biomembranes with their lipophilic moiety and bind to surface glycoproteins and glycolipids. Most terpenoids are lipophilic in nature and readily interact with the lipophilic inner core of membrane bilayers [37]. Flavonoids inhibit the influx of L-glutamine and myoinositol into P. falciparum-infected erythrocytes [48]. These phytochemical constituents may inhibit parasite growth and multiplication, resulting in a reduction in parasitemia and body temperature.
We found that the aqueous fruit extract of T. arjuna presented a group of flavonoids, terpenoids, alkaloids, tannins, and saponins. Our results are consistent with those of previous reports of the chemical constituents of T. arjuna [49,50]. Secondary metabolites, particularly flavonoids, alkaloids, tannins, and saponins, are protective against Plasmodium parasites [44][45][46][47]. The most abundant compounds in the fruit extract of T. arjuna were pyrogallol, gallic acid, shikimic acid, oleamide, 5-hydroxymethylfurfural,1,1-diethoxy-ethane, quinic acid, and furfural. The antimalarial activity of this extract may be attributed to the synergistic effects of these compounds. Interestingly, the identified compounds from the fruit extract of T. arjuna including pyrogallol, gallic acid, shikimic acid, cinnamic acid, and quinic acid are interrelated via biosynthesis pathway [29][30][31]. Since, most nonvolatiles will decompose at between 400 and 1000 °C [51]. Therefore, the identified compounds which non-volatiles cannot be vaporized and decomposed easily. In addition, in this study, the injector temperature of GC-MS was set at a constant of 250 °C, and the maximum temperature of the oven was set at 300 °C. So, this thermal condition inapplicable for decomposition process.
The bioactivities of the two major compounds, shikimic acid and 5-hydroxymethylfurfural, further explains why the T. arjuna extracts significantly